1. Field of the Invention
This invention relates to the quantitative separation and detection of organic molecules. More particularly this invention relates to systems for separating a species of organic molecules, and for detecting and quantifying the separated molecular species with a continuous wave laser through which the separated organic molecular species is conveyed.
2. Description of the Prior Art
An extensive range of both industrial and clinical processes are based on determination of the identities and concentrations of organic molecules in solution. Primary samples obtained for analysis may range from industrial fermentation processes to the bodily fluids of animals or humans. These samples typically contain a very wide range of molecules and molecular complexes in an equally wide range of concentrations. To deal with this a typical assay system will consist of six fundamental parts: sample collection, sample filtration, molecular separation, molecular recognition, concentration measurement, and data analysis and presentation. Not all assays require all six steps; for example, filtration is often not required. However, separation, recognition, and concentration measurement are usually in the critical path to data analysis. The present invention provides a novel and greatly improved methodology to this critical path activity.
Chemical and biochemical separation technologies have been extensively developed over the past 100 years. A complete review of all separation technologies is beyond the scope of this application. However, it is useful to review some of the most common separation technologies and to compare and contrast them to the invention disclosed here.
Two of the most common separation technologies in current application are electrophoresis and chromatography. Electrophoresis refers to the migration of charged molecules or ions in a liquid medium under the influence of an applied electric field. This is also known as electroosmosis. Chromatographic separations operate by introducing a sample into a flowing stream of gas or liquid that passes through a bed of support particles. The support particles are known as the stationary phase and the flowing stream is known as the mobile phase. The driving force for chromatic separations may be fluid or gas pressure, gravity, or capillary action.
Both electrophoresis and chromatography have many embodiments. Electrophoretic separations are used to analyze and characterize proteins and nucleic acids, especially DNA and RNA. Chromatography is often used to isolate specific molecules and characterize smaller molecules such as drugs and amino acids. Although the inventions described in this disclosure can be used in both types of separations, they find greater application in electrophoresis than chromatography. Therefore we will describe present electrophoretic practice in greater detail.
The first electrophoresis method used in the study of proteins was the moving boundary method devised by Tiselius in 1937 which was a fully fluid electrophoretic system. Presently, most electrophoretic separations are carried out in a porous support medium such as cellulose paper, agarose, and polyacrylamide gel films. This is called zone electrophoresis. A related technique called capillary electrophoresis uses electroosmotic flow separation in 50-100 .mu.m capillary placed in very strong electric fields to separate molecules, with or without the use of a porous matrix.
Molecular electrophoretic separation technology used in practice today takes advantage of separation by both electroosmosis and molecular sieving. Electroosmosis alone may be used to separate biomolecules. However, electroosmotic separations require longer separation distances--up to a meter or more--and additionally require 10-20 kilovolt potentials along the electrophoretic length. Presently, only capillary electrophoresis is performed this way. In slab geometries, meter long separations are impractical, and 20 kilovolt potentials can lead to excessive heating and electronic arcing problems. Therefore, gels are introduced to act as molecular sieves to enhance molecular separations. Gels provide quicker separations in smaller distances and use lower voltages. Almost all molecular sieves used in separations are gel polymerized molecules from poured or cast solutions. Sieve pore sizes may range from 0.5 .mu.m in agarose gels (designed to separate large DNA fragments) to 0.01 .mu.m for polyacrylamide gels used to separate standard protein mixtures.
The rate of migration of a charged particle in a solution is a function of five variables. These include 1) the net electrical charge of the molecule (which is a function of the pH of the buffer solution), 2) the size and shape of the molecule, 3) the electric field strength, 4) properties of the support medium (e.g. pore size and fixed charges), and 5) the temperature of operation.
The net electrical charge on a molecule is a strong function of the pH of the buffer medium. The separation method known as isoelectric focussing separates amphophilic molecules (molecules possessing both positive and negative charges) such as proteins, by their migration in stable pH gradient. The protein moves to the zone in the medium where the pH is equal to the isoelectric point of the protein.
As opposed to isoelectric focusing, most electrophoretic procedures result in the separation of molecules by molecular weight. Under these circumstances, separation is accomplished by the sieving properties of the gel support medium or matrix. Smaller molecules move faster than larger molecules in appropriately sized gels. To aid in providing a strict molecular weight separation, protein molecules are often denatured in a detergent such as sodium dodecyl sulfate (SDS) to remove the effects of tertiary and quaternary structure on molecular mobility. For separation of standard proteins of molecular weight from 200,000 to 10,000 daltons a 7.5% polyacrylamide gel may be used to provide a pore size down to 5 nanometers (50 Angstroms). Most gel systems rarely resolve more than 100 bands of protein.
In order to enhance the number of electrophoretically resolvable bands, O'Farrell in 1975 introduced the method of two-dimensional (2D) electrophoresis. In this technique charge dependent isoelectric focussing is used in the first separation axis. After separation, the one-dimensional gel is extruded from the gel tube and placed in contact with a thin polyacrylamide gel slab that incorporates SDS. At the end of the process the polypeptides are detected, usually by chemical staining or autoradiography. 2D gels allow the simultaneous visual detection of more than 1000 peptides. Thus they are often used in cell or organ "fingerprinting" applications.
Immunologic techniques are often combined with electrophoretic separations to provide powerful diagnostic tools. In immunoelectrophoresis, nondenatured proteins are separated on an agarose gel. The method of applying the antibody to the separated mixture often determines the class of immunoelectrophoretic technique being used. In standard immunoelectrophoresis, the antibody is applied to a trough next to the separation medium and both antigens and antibodies are allowed to diffuse into each other, forming arcs of immunoprecipitates. In crossed immunoelectrophoresis, a second dimension of electrophoresis is used to drive the antigens into an antibody coated gel, while in immunofixation, antibody is spread over the gel and nonprecipitated proteins are washed away.
Often the amount of antigen present in a separation medium is insufficient for detection in a gel matrix. To overcome this limitation proteins are often transferred to a solid phase such as nitrocellulose paper where either radioactive isotopes or enzymatic assays of greater sensitivity can be applied. This procedure is called Western blotting.
Small, single stranded DNA fragments are separated under denaturing conditions on polyacrylamide gels. Larger, double stranded molecules can be separated in more porous agarose gels. However, duplex DNA longer than 20 kilobase pairs cannot be separated in a size-dependent manner in a one-directional electric field. This is related to the way the long DNA molecules move through the pores of the gel. They are probably elongated in the direction of the field, and may wrap around the agarose gel matrix and become immobilized. Thus the DNA molecules must be made to continually reorient if they are to move through the gel. To accomplish reorientation, pulse-field electrophoresis has been developed. Here two nonhomogeneous, mostly perpendicular fields are applied to the separating DNA mixture. One field causes the separation while the other promotes reorientation. The newest systems optimize the angle between the two fields. Pulse times are generally of the order of a minute. To separate large DNA fragments, gel pore sizes of up to 0.2-0.5 .mu.m are used.
Capillary electrophoresis is a recent variation on the traditional slab gel electrophoretic techniques. Here many of the standard techniques such as zone electrophoresis, isoelectric focussing, and gel electrophoresis are carried out in 25-75 .mu.m fused silica capillary about 100 cm long. The capillary is connected to a high voltage supply on one end and a detector and/or fraction connector on the other end. The advantages of performing electrophoretic separations in capillaries include improved heat dissipation (which allows use of steeper potential gradients and hence, faster separations), reduced sample volume requirements, reduced zone (or protein band) broadening, and easier process automation. Sample volumes are kept in the picoliter or nanoliter range in order to maintain the buffer-electrolyte to solute concentration ratio of at least 10.sup.3, which minimizes distortion in the applied field caused by the presence of the sample.
Many detection schemes are possible for detection of the electrophoresing sample at the end of the capillary tube. These include optical absorption and fluorescence, radiometric, and mass spectrometer means. With the most sensitive methods as little as 10.sup.-20 moles of substance can be detected.
Most recently it has been shown by groups at the University of New Mexico (UNM) and at Ciba-Geigy Labs that it is possible to perform electroosmotic amino acid separations in lithographically patterned channels etched into a glass substrate. These channels function in a similar manner to capillaries used in electrophoresis. The UNM micro-separations were extremely rapid (as short as 4 seconds) and required little material. This is the first step toward building full capillary-type electrophoretic separation systems on a planar substrate, or chip. Attempts are now being made to form miniature, multistage separation and analysis systems using semiconductor technology, that incorporate electrically active substrates for separation channel switching. However, there are limitations in performing electroosmotic separations on active semiconductor substrates. Electrical potential differences in the multikilovolt range are extremely difficult to maintain in integrated circuits. In addition, meter long separations required for large biomolecule separations demand large planar areas, even if spiral winding of the channel is employed.
All existing electrophoretic systems have limitations and drawbacks which we will now discuss. Identification of the separated molecules is a major issue in most applications of electrophoresis. There are two basic types of molecular identification systems in use. In one case the electrophoretic process is stopped while all molecules are still in the electrophoretic medium resulting in the formation of an electrophoretogram. The separated, arrayed molecules are detected in parallel by chemical staining to reveal their presence, or by measuring a physical property of the molecules, such as fluorescence or the effects from radioactive labelling. This type of detection system is common in protein and DNA slab gel electrophoresis.
In the other system the molecules are detected as they sequentially exit the electrophoretic system. Detection may be by optical absorption, fluorescence, or radioactivity. This method is normally used to detect the results of capillary electrophoretic separations.
The Western blot methodology described above is a hybrid of these two methods, where the proteins of interest are transferred from the electrophoretogram to another solid support for readout.
Key issues in all detection systems include the dynamic range, sensitivity, specificity, calibration, and time to result. These issues will be examined in order here.
Electrophoretic systems separate molecules by their unique directed motions in an electric field. It is often desired to measure the quantity of a rare molecule which has drifted a distance extremely close to that of a substantially more abundant molecule. Methods to accomplish this include 2-dimensional electrophoresis (described above), use of a specific label (such as an antibody), or isolating the desired region of the gel or liquid fraction and reelectrophoresing this portion of the sample. To sharpen the electrophoretic zone structure, thinner gels, longer electrophoretic paths, and steeper voltage gradients are resorted to. As yet there has been no attempt to provide active feedback to sharpen electrophoretic bands.
Another challenge is to find the appropriate gel concentration to resolve both high molecular weight (&gt;500,000 daltons) and low molecular weight components on a single separation. This may be dealt with by application of a discontinuous gel with two or more concentrations, or by use of a gradient gel system. Again, there has been no attempt reported to provide active, separate modulation of the high and low molecular weight regions after separation.
Sensitivity is a strong function of the stain used to report the presence of macromolecules. Clearly fluorescence staining with specific ligands is substantially more sensitive than standard absorption stains (such as Coomassie Brilliant Blue). However, fluorescent detection systems (which often require an ultraviolet or argon ion laser excitation source) are generally more expensive and slower in readout of electrophoretograms. Typical slab gel systems operate with several hundred micrograms of sample material. Capillary electrophoretic systems can operate with nanograms of sample. Detection thresholds can be exceptionally low if fluorescent or radioactive stains are used. Amounts as low as 10.sup.-20 moles can be detected in a given eluted fraction. Absorption measurements are usually carried out in the ultraviolet region, where proteins and nucleic acids are strongly absorptive. It is desirable to have a detector system which uses an inexpensive, long lived light source, and which has sensitivity in the sub-nanogram range.
Most gel systems are cast or poured from a solution. Variations in component concentrations, room temperature, buffer strengths, etc., can lead to variable drift rates. In slab gels the solvent front often does not move uniformly, creating a variation in separation distances across the slab or distorted bands within the gel. This can arise from excessive drying of the gel or from overapplication of the sample. In capillary electrophoresis, capillary bore variations as well as variations in the above mentioned parameters will create variations in elution time. System calibration is possible either by adding monitor substances to the elution channel, or by providing a separate lane for electrophoresing standard molecules of known molecular weight. Calibration can be enhanced with automated densitometric readout to provide curve fitting of lane electrophoretic separations and digital processing of electrophoretograms to remove run time variations. Nevertheless, any approach that will enhance reproducibility represents a major advance.
Many electrophoretic separations require 1 hour or longer. To this must be added gel preparation time, stain time, and readout time. An entire separation procedure may require 4-6 hours from start to finish. Additional time is required if enhanced resolution is needed, due to the use of longer gels and longer run times. Electrophoresis can be a rate limiting step in data delivery in both research and clinical applications. It would be highly desirable to have an electrophoretic medium that is reusable, precalibrated, and permits higher resolution, while providing shorter run times, active readout, and good end point detection. Reduction of the entire process to less than 30 minutes is a highly desirable goal in many areas of clinical practice, as results could be provided during the patient's diagnostic visit.
Quantitative molecular detection is a also key part of any chemical assay system. Electrochemical means may be employed using ion specific electrodes or biosensors to measure molecular concentrations directly. Such devices exhibit micromolar sensitivity, and are available for a limited number of molecules.
Direct optical detection relies upon fluorescence or absorption measurements; concentration is assessed by the strength of the signal. Not all molecules of interest have an adequate direct optical signature, however. Concentrations of macromolecules such as enzymes in solution (e.g. blood plasma) are difficult to measure directly this way.
A broader methodology of detection involves the use of a so-called "reporter" molecule. In this method the molecule of interest is brought into contact with a reagent which binds to the molecule and provides its own measurable signature. One such signature is a change in the optical absorbance of the reporter molecule. The rate of change of this signal is directly related to the concentration of the molecule. A wide range of such spectrophotometric systems have been developed and sold. Concentrations in the nanomolar range can be measured this way. Careful control of reagent concentration and purity is required to obtain accurate results.
The reporter molecule is sometimes a radioactive "tag" attached to the molecule of interest. Concentration is derived directly from the strength of the radioactive signal. Proper storage, handling, and disposal of the requisite radioactive materials is a general area of concern in the health care field.
In some cases fluorescent tags may be attached instead. Such tags usually require ultraviolet excitation. Some molecules are unstable under such actinic irradiation so this method is not universally applicable.
A new class of research instrument has appeared on the market which measures the real-time binding kinetics of label-free biomolecules. These systems all use the behavior of light reflection at the near grazing angle boundary of different refractive indices as a detection modality when the molecule of interest binds to a specific ligand attached to the optical surface. This phenomenon is known as frustrated total internal reflection (FTIR), and systems based on it have demonstrated an ability to detect molecular concentrations approaching the nanomolar level in some test conditions.